The examination of foetal cells for early detection of foetal diseases and genetic abnormalities is carried out in connection with many pregnancies, in particular when the maternal age is high (35 years or above) or where genetic diseases are known in the family. Foetal cells may be obtained by amniocentesis, the removal of amniotic fluid from the amniotic cavity within the amniotic sac or by chorion biopsy, where biopsies are taken from the placenta, so-called invasive sampling.
Prenatal aneuploidy screening employs either standard chromosome analysis or FISH analysis using specific DNA probes for the elucidation of numerical aberrations of chromosomes, particular chromosomes 13, 18, 21, X and Y in the foetus.
Due to the invasiveness of the methods described above and the risk of abortion associated herewith, it would be advantageously to perform foetal diagnosis by a non-invasive procedure, such as for example by use of a maternal blood sample.
During pregnancy a variety of cell types of foetal origin cross the placenta and circulate within maternal peripheral blood. The feasibility of using foetal cells in the maternal circulation for diagnostic purposes has been hindered by the fact that foetal cells are present in maternal blood in only very limited numbers, reported numbers have been from one foetal cell per 105-108 nucleated maternal cells or 1-10 foetal cells per ml maternal blood.
In order to use foetal cell present in maternal blood for diagnostic purposes, methods suitable for isolation and/or identification of foetal cells are required.
Most foetal cells cannot be distinguished from maternal cells on the basis of morphology alone, thus alternative methods of identification of foetal cells have been investigated.
Foetal cells present in maternal blood include erythroblast, foetal leukocytes and trophoblast cells. Recently, foetal cells with stem cell properties have been indicated to be present in pregnant women and described as pregnancy-associated progenitor cells (PAPCs) and foetal mesenchymal stem cells (Khosrotehrani and Bianchi, 2005)
As described above different types of foetal cells, such as nucleated erythrocytes have be identified in maternal blood samples but so far, the efficiency of detecting these cells is very low. This may be due to the low number of foetal cells present in a maternal blood sample and/or due to the method employed for detection of said foetal cells.
Due to the very limited number of foetal cells in maternal blood separation or enrichment of the maternal blood sample with respect to the foetal cells is often conducted by for example negative selection, i.e. removal of maternal cells.
Maternal cells may be removed by density gradient centrifugation, by removing maternal cells with an antibody to a cell surface antigen or alternatively by lyses of maternal erythrocytes, optionally combined with immunologic methods for removing the maternal cells.
Alternatively foetal cells are separated from maternal cells by using flow cytometric methods or alternatively by a new technique, in which, a two-step enrichment procedure is performed to isolate trophoblast cells, consisting of a density gradient centrifugation and negative immunoaffinity isolation using Magnetic Activated Cell Sorter (MACS) Separation techniques and monoclonal antibodies or ligands specific to foetal cells.
It is however, a problem that due to the enrichment/separation procedures some of the foetal cells may also be removed leading to even fewer foetal cells in the blood sample to be analysed. Particular antibody selection methods may lead to a loss of foetal cells not expressing the antigen at a sufficient level.
Most methods are thus biased by the selection criteria of the enrichment/separation step. Two methods for unbiased quantification of total amount of foetal cells are available. Both methods are based on detection of Y-chromosome sequences. One is quantitative PCR, which is rather imprecise and the other is FISH staining of Y-chromosome in nuclei followed by counting. The enrichment step in the latter is based on initial carnoid fixation of whole blood, which dissolves all cell membranes thereby lysing the erythrocytes and removing the cytoplasm and many nucleoproteins of the nucleated blood cells. This treatment thus results in a nuclear pellet, which can be smeared onto slides. These slides are ideal for FISH-analysis, since the removal of so many proteins makes the nuclear Y-chromosome sequences easy assessable for the Y-chromosome specific probe. Both the PCR and FISH based techniques have been used to quantify total number of foetal cells and the most reliable reports all come to similar results, namely about two foetal cells pr. ml full blood. The problem with this quantification is however, that neither of the two methods allows identification or characterisation of the foetal cell disclosed. For the PCR-method due to only specific Y-chromosomes sequences being measured, and for the FISH-method due to the carnoid fixation removing all cytoplasm.
Immunocytochemical methods for detecting erythroblast, lymphoblast and trophoblast have been developed and blood from pregnant women investigated. In general very varying results have been obtained, some very compatible with the previous mentioned cell unspecific estimates. If however the foetal origin of a candidate cell is established without doubt (double and independent verifications) the general experience is, that the number of the three candidate cell types mentioned are one to two orders of magnitude lower than what was found by the cell unspecific methods. Thus the maternal blood may comprise foetal cells which are none of these three proposed cell types.
In general the methods described so far are laborious and complex and thus not suitable for routine analysis.
Thus improved methods for identification of foetal cells are required in order to utilise foetal cells circulating in pregnant women for non-invasive pre-natal diagnosis.
Usable tools in elucidating the identity of foetal cells are binding members such as antibodies or antibody fragments or alternative binding molecules capable of specifically recognising said foetal cells.
In general antibodies are generated by immunization of animals, preferably rodents. This usually requires a substantial amount of the antigenic material for sequential immunisation and/or several rounds of selection. These methods are thus not suitable when only a small amount of the antigenic material is available and when the antigenic material comprises multiple independent antigens. It is further normally accepted that a high purity of the target antigenic material is required to obtain suitable antibodies using these methods.
In some situations a binding member towards a minor fraction of molecules present in a sample is desired. That may be such as a specific cell type in a sample comprising multiple cell types. As purification of this minor fraction of molecules for which a binding member is thought is not feasible or otherwise undesired, alternative methods are required for generation of binding members towards target antigenic material present in a low ratio compared to the amount of non target antigenic material in a sample.
As described above the abundance of foetal cells in maternal blood is very limited and only available in small amounts and the ratio of foetal cells compared to maternal cells in maternal blood is less than 1/1000. In addition foetal cells are not easily purified from the maternal blood sample, as a substantial amount of the foetal cells may be lost during purification, thus conventional methods of generating antibodies are not efficient for the purpose of generating antibodies for recognition of foetal cells.
As described herein, the applicant has developed a method of identifying foetal cells furthermore said method allow the generation of binding members recognising said foetal cells. Hereby tools suitable for the development of prenatal diagnostic methods are made available.
It is further foreseen that the method of generating binding members may have a general application for generation of binding members recognising target antigenic material of limited availability or for the generation of binding members recognising target antigenic material present intermixed with non-target antigenic material in a sample.